Biology

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This is the collection for the University of Waterloo's Department of Biology.

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Test Submission 1
(2024) Carmini, Priscilla
test abstract
Wastewater Surveillance of Influenza (A, B) and Respiratory Syncytial Virus (RSV) in Southern Ontario
(University of Waterloo, 2024-07-16) Abu Farah, Joud
Wastewater-based surveillance (WBS) of SARS-CoV-2 has been highly effective at tracking trends of COVID-19 infections across the globe. This success led to the question of the possible applicability of WBS in monitoring other respiratory viruses that are also posing a concern for healthcare systems. Traditional influenza and RSV surveillance consists of monitoring hospital admissions, clinical testing data and outpatient visits. Even when clinical surveillance and hospitalization admissions are used to monitor the spread of these infections, they are often underestimated, with a lag between detection and community spread. In this study, wastewater surveillance of influenza A, influenza B and respiratory syncytial virus (RSV) was investigated, by quantifying the viral RNA of these viruses in wastewater and comparing these trends to clinical metrices. The solid–liquid partitioning behaviors of influenza A, influenza B, RSV and SARS-CoV-2 in wastewater were examined to understand the behavior of the viruses in wastewater. Analyzing whether each virus is present in the liquid or solid fraction of wastewater may affect the data interpretation and inform further method development. The viral RNA in the liquid and solid fractions were separated and enriched by undergoing different centrifugation settings, overnight polyethylene glycol (PEG) precipitation followed by centrifugation, or ultrafiltration using a Centricon Plus-70 device. The influenza A, influenza B and RSV viral RNA concentrations were found to almost exclusively partition in the solids fraction of wastewater, which increased with an increase in centrifugal settings, unlike SARS-CoV-2 that had a more even split in signal between the solids and liquid fractions. The effectiveness of normalization using the endogenous pepper mild mottled virus (PMMoV) was also examined, by comparing the normalized influenza signal with the raw signal. Normalization using the PMMoV biomarker did not increase or hinder the correlation with clinical testing data relative to the raw influenza signal in wastewater. Wastewater samples were collected once weekly from two wastewater treatment plants in the Region of Waterloo, Canada, from September 15, 2022, to June 21, 2023. Wastewater was treated overnight with polyethylene glycol (PEG), centrifuged (12,000g 1.5 h), then extracted (pellet) with Qiagen RNeasy PowerMicrobiome Kits and quantified using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). The RNA concentrations of influenza A, influenza B and RSV in wastewater strongly correlated with the cases reported by episode date and hospitalizations, in the cities of Kitchener and Waterloo (Spearman’s Rank correlation coefficient rho ranging from 0.41-0.85). Wastewater surveillance can be a very effective additional surveillance tool to support public health officials in monitoring the trends of respiratory viruses in communities. Further development of WBS for respiratory virus monitoring will help public health to better prepare for these and other emerging pathogens in the future.
Degradation of Polyethylene Terephthalate (PET) and Polyamide (PA)
(University of Waterloo, 2024-07-16) Griffiths, Erin
Microplastics have become an increasing concern to humans and ecosystems as plastic production continues to soar, due to their prevalence in the environment and lifespan. Plastic is cheap and durable making it an ideal industrial and commercial material. However, because of this popularity, it resides in most places on earth, including in human blood, and is difficult to remove due to its small size. These plastics can enter the environment through numerous methods, from landfills and dumps to washing machines and sinks. In recent years, there has been significant investigation in reducing plastic pollution. This a difficult task attributed to the varying size, shape, polymer type, chemical properties and location plastic can be found. It’s critical to understand the rate of degradation and the factors that influence it for two main reasons; it provides accurate timelines of degradation and techniques that may increase degradation need a starting point. In Chapter 2, I investigate the degradation rate of laboratory grade polyethylene terephthalate (PET) using a model enzyme (Huimcola insolens cutinase) to hydrolyze the plastic. This research aims to characterize the polymers used such that results can be compared and identify the analyses which capture degradation and characterize the polymer best. Environmental factors controlling enzymatic plastic degradation are not well studied and this experiment aimed to study the effect of incubation temperature, exposure to freeze-thaw cycles (FTCs) and extreme temperatures on the degradability of laboratory-grade PET. In addition, we also assessed the degradability of consumer-grade PET, sourced from plastic bottles, for comparison to the laboratory-grade PET. The first test was under variable temperatures, where plastic was incubated at 25 ˚C, 40 ˚C and 55 ˚C. The results show increased temperatures increase the rate of polymer degradation. The second set of tests were conducted under different pretreatments; treatments the plastic would undergo before incubation at 40 ˚C. Plastic was exposed to a series of freeze-thaw cycles (FTCs) or extreme temperatures (-70 ˚C or +55 ˚C). It was found any type of pretreatment increased the rate of degradation compared to plastics that did not undergo any pretreatment. The final condition tested was plastic type, where PET water bottles were obtained and incubated at 55 ˚C to determine the differences in degradability between laboratory-grade PET and consumer-grade PET. Consumer-grade PET was found to not have any significant degradation after 10 weeks of enzyme exposure, raising serious concerns regarding its degradability and lifespan. This result suggests that modifications to the consumer-grade PET during the fabrication process, such as heat treatments, are altering its chemistry and its degradation kinetics. Analyses for degradation and characterizing the polymers included: Fourier-transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and tensile strength measurements. Analysis of the crystallinity, tensile strength, SEM images and FTIR spectra measured indicate that PET’s physical and chemical properties were modified when degraded. Overall, the PET’s tensile strength decreased and the crystallinity increased with increasing hydrolytic degradation. FTIR spectral changes were seen early on, with peaks of interest at 1237 cm-1, 1016 cm-1 and 1087 cm-1, and finally at 1716 cm-1, and the flattening of these peaks increased with increasing hydrolytic degradation. The results highlight that enzymatic degradation rates can be highly variable due to differences in environmental conditions. It also highlights the large difference in the degradability of consumer-grade versus laboratory-grade PET, which has significant implications for in situ environmental degradation rates. In Chapter 3, I investigated the rate of laboratory-grade PET and polyamide (PA) degradation in stormwater pond sediment over a 16 month period in a stormwater pond in Kitchener, Ontario. Microplastic accumulation in the environment, especially in bodies of water and sediment is a well-known problem. Stormwater ponds act as a microplastic sink and draw pollutants from urban and industrial wastewater before it enters oceans or lakes. This results in high levels of microplastics remaining in stormwater pond sediment. Stormwater ponds are an excellent site to determine realistic plastic degradation in the environment, in a contained area where high concentrations of plastic is known to be present. To date, no long-term polymer degradation studies have been conducted in a stormwater pond despite the rising popularity of these ponds. For this study, 8 pore water samplers (peepers) were packed with pond sediment and plastic pieces were inserted into each cell of the peeper. An additional 8 peepers filled with water, such that pore water chemistry could be collected. The peepers were inserted into the pond sediment and sacrificed periodically over the course of 16 months. For the first 8 months both PET and PA plastic increased in mass as they absorbed water. After 16 months of field incubation, PA had degraded by 0.42% and PET was still net positive (higher mass than before the incubation) however it was close to its original weight. The obtained results highlight the lack of degradation to plastics in stormwater pond sediment and suggest lifespans are longer than previously estimated. Based on previous degradation studies under sediment conditions, this study suggests that stormwater pond sediment is the least effective at degradation polymers, which may be attributed to the pond water chemistry and microbial communities present. Microplastics are known to accumulate in stormwater pond sediment but they are found to degrade at slower rates than other sediment profiles. The laboratory experiment results in Chapter 2 show under ideal conditions laboratory-grade PET degrades minimally at low temperatures. Additionally, the lack of degradation seen with the consumer-grade PET in Chapter 2 suggests that under environmental conditions, the polymer would take even longer than the laboratory-grade polymers to degrade. The combination of Chapter 2 and 3 demonstrate the difference between ideal and environmental conditions for polymer degradation. This research provides evidence to strongly advocate for the removal of microplastics before they enter the environment as I have proven they take considerable lengths of time to degrade under various conditions. I encourage this research to be used by any future researchers who hope to develop methods for plastic pollution reductions.
Patterns in stream biofilm communities and organic matter processing in an agricultural stream network: A multi-scale assessment of the influence of groundwater
(University of Waterloo, 2024-06-03) Banks, Lauren
Benthic stream biofilm communities support stream ecosystem structure and function by mediating nutrient and carbon cycling. Understanding how environmental factors shape biofilm communities in stream ecosystems is therefore essential. Biofilm communities have been shown to be strongly influenced by nutrient availability and temperature, factors that can be modified by groundwater input at multiple spatial scales. However, in enriched streams, groundwater input as a driver of heterogeneity in surface water environmental conditions has not been well-explored among stream reaches (kilometer scale), habitat types (meter scale), and patches (centimeter scale), nor has the seasonal consistency of these relationships been studied. To investigate the association of groundwater input to biofilm communities, I conducted three interconnected field studies in Kintore Creek, a nutrient-rich agricultural stream network in Ontario, Canada. First, I assessed if variability in groundwater input altered patterns of biofilm communities and cellulose decomposition among reaches over four temperate seasons (Chapter 2). Next, I compared habitats (i.e., riffles and runs) in reaches with high, moderate, and low groundwater inputs to determine if habitat type modified the effects of groundwater input on stream biofilm communities and cellulose decomposition by varying environmental conditions (Chapter 3). Lastly, I assessed the response of stream biofilm communities and cellulose decomposition to a gradient of groundwater upwelling at the patch scale and tested whether small scale variations in environmental conditions are associated with biofilm communities and cellulose decomposition (Chapter 4). The results of Chapter 2 showed no within season association of groundwater input to biofilm communities, with vii seasonality driving heterogeneity in biofilm communities. Findings in Chapter 3 demonstrated that habitat type modified effects of groundwater input on biofilm communities. Groundwater influence was expressed by greater primary production and decomposition in runs in reaches with groundwater input compared to runs in the reach with no groundwater input. At the patch scale (Chapter 4), groundwater upwelling did not appear to generate substantial variation in surface water conditions, and variability stream velocity was the primary driver of heterogeneity in stream biofilm communities. The findings of this this thesis are in contrast to past work that found effects of groundwater on stream biofilm communities in nutrient-poor streams. These results may be due to cumulative effects of groundwater input throughout the stream network, thereby limiting the ability to detect environmental drivers of groundwater influence at small spatial (i.e., habitat, patch) scales. Therefore, additional studies comparing catchments with differing levels of groundwater are needed to fully understand the influence of groundwater on stream biofilm communities in differing landscape contexts. A major challenge across spatial scales was the ability to represent the impact of groundwater inputs through environmental measures and biofilm communities, suggesting further investigations at the stream water – biofilm interface is required to disentangle the environmental drivers associated to heterogeneity in biofilm communities. The results of this thesis suggest that the influence of groundwater input on stream biofilm communities and processes depends on the context of stream ecosystem, therefore understanding effects of groundwater input requires future research across a diverse range of stream ecosystems.
The Development and Evaluation of Next-Generation Metallic Nanomedicines for Oncology
(University of Waterloo, 2024-05-27) Youden, Brian
Nanoparticles (NPs) are ultrasmall objects with profound applications in research, industry, and medicine. Next-generation nanomedicines, such as gold, hafnium, iron, and copper nanoparticles, are particularly interesting due to their excellent physical, chemical, and quantum properties that can be exploited for cancer diagnosis and therapy. However, despite their demonstrated preclinical effectiveness, the potential of these inorganic nanomedicines, both in oncology and the broader medical field, is hampered by mechanistic uncertainty and a lack of detailed regulatory guidance. Together, these factors have resulted in many failed clinical trials and unexpected and sometimes severe side effects for approved formulations. The therapeutic efficacy and toxicity of nanomedicines are controlled by an extremely complex interplay of nanoparticle physicochemical properties and individual patient biology, where many confounding factors exist. This makes designing and evaluating nanomedicines a challenging task. To progress metal-based nanomedicines to the clinic and for them to be considered safe, even in the life-or-death circumstances of cancer, a deep understanding of nano-bio interactions is necessary across different stakeholders. This includes physicians, academia, industry, and government. By understanding and utilizing these in vivo behaviors, powerful nanomedicines and novel treatments can be applied to oncology. This thesis begins with a summary of the fundamental concepts relating to nanotechnology and the origins, properties, and treatment of cancer. Chapter 2 expands this discussion for a comprehensive analysis of cancer nanomedicines and their structure-activity relationships (SARs) in the body, which are central to both treatment efficacy and safety. Fundamentally, SARs describe the interactions between NP properties and the biological systems that ultimately produce their effects. To assist in the communication of this information, identified SARs were integrated into a simple, adaptable, and guiding framework composed of a parameter space, a pathway model, and various evaluation metrics. By resolving the complexity of nanomedicine into three parts, representing the interactions of NPs with 1) whole organs, 2) individual cells, and 3) fundamental biochemical pathways, this framework provides a clear illustration of how to fine-tune nanomedicines via pathway analysis. This framework and SARs were then used to guide the design, application, and evaluation of next-generation nanomedicines containing gold and copper. Gold nanoparticles (GNPs) have long been proposed as promising agents for cancer phototherapy and image-guided radiation therapy (IGRT) due to their strong absorption of near-infrared (NIR) light and X-rays. GNPs are also among the most studied NPs owing to their general biocompatibility and easy synthesis. Despite this, only one GNP has been approved for clinical use owing to long-term safety concerns. Among various SARs, those related to size are often the most critical parameters for both efficacy and safety. This stems from both the nanoparticles themselves and the size-restrictive nature of kidney, liver, and tumor filtration of blood. To optimize the use of GNPs for enhanced IGRT, drug delivery, and photothermal therapy (PTT), drug-loadable lipid NPs were utilized as a scaffold for GNP assembly, forming a versatile nanocomposite (Lipogold). Overall, this allows small NPs to function collectively as one larger nanoshell with plasmonic properties. Over time, this shell will degrade as the soft liposome core is stressed and deformed, resulting in renal-clearable NPs that can be cleared by the body following treatment. This thin shell of gold also optimizes the Auger process for RT and enables PTT, while the hollow core allows for encapsulation and delivery of drugs and molecular contrast agents. Thus, Lipogold nanocomposites demonstrate the advantages of both large and small NPs while adding multifunctionality. In this work (Chapter 3), medical radiation sources and cellular models were used to test their ability to sensitize cancer cells to megavoltage X-ray radiation therapy, provide contrast for computed tomographic (CT) imaging, deliver drugs, and engage in NIR-based PTT. In addition to GNPs, plasmonic copper sulfide (Cu2-XS or just CuS) NPs are also emerging as increasingly popular nanomedicine candidates due to their photothermal properties, biodegradability, an ability to convert less-toxic H2O2 into more potent reactive oxygen species (ROS) for chemodynamic therapy (CDT). However, this approach in cancer therapy is fundamentally limited by several factors, principally the low concentration of H2O2 in the body. To overcome this issue, the properties of the tumor microenvironment (TME) were exploited for nanomedicine design, where CuS NPs were combined with the enzyme glucose oxidase (Gox) for a synergistic combination of starvation therapy, CDT, and PTT. Gox was used to convert glucose, which is upregulated in the TME, into H2O2 and acid, starving the cancerous cells and activating the Fenton-like reactivity of the CuS NPs. Deep-penetrating NIR could then be used for PTT and to enhance reaction kinetics specifically at the tumor site. The fundamental reaction mechanism was also investigated, highlighting the accelerative effect of chloride ions on the copper-Fenton reaction, which are present at high concentrations within skin and individual cells. In Chapter 4, the therapeutic efficacy and biocompatibility of the Gox@CuS nanocomposite were demonstrated using in vitro and in vivo melanoma models. To further improve the safety profile of the Gox@CuS nanocomposite, the emerging technology of microneedle patches were explored as a transdermal drug delivery approach. Since conventional injections can lead to off-target uptake and toxicity, transdermal delivery may improve both efficacy and safety by maximizing local delivery and limiting blood exposure. This approach was extensively reviewed (Chapter 5) to determine the viability, design considerations, and fabrication methods of MNs containing light-responsive NPs such as Gox@CuS. Applications outside oncology were also reviewed to fully understand the advantages and limitations of this delivery system. Gox@CuS were then integrated into dissolvable polymeric microneedle (DPMN) patches and compared to hypodermic injection using another mouse melanoma model. In this study (Chapter 6), the microneedle patches were demonstrated to deliver a higher amount of Gox@CuS to the tumor site and reduce the risk of systemic toxicity. Further mechanistic insight into the catalytic behavior of CuS NPs was also collected, specifically identifying the effect of chloride ions on the generation of both hydroxyl radicals and singlet oxygen. Overall, this thesis contributes to our overall understanding of cancer nanomedicine and demonstrates several novel next-generation treatment strategies using metal-containing NPs. The framework proposed in this work is an adaptable and potentially valuable resource for researchers and regulators to understand SARs. Additionally, the pathway modelling used by this framework can assist in the development and integration of machine learning models that will increasingly play a role in the regulatory and industrial development of nanomedicine formulations.
Differential Gene Expression Analysis using Resampling
(University of Waterloo, 2024-05-09) Yang, Yifan
The primary objective in high-throughput sequencing is to identify differentially expressed genes, which provides substantial information of gene expression and regulation. A persistent challenge in this field is the bias caused by limited replicates. To address this, we have developed a novel Bootstrapping method. This approach enhances the power of DEG detection by augmenting the sample data points. New data points are generated through weighted geometric combinations of bootstrap samples and a pooled distribution. The pooled distribution consists of sample data from genes with similar expression levels. Through simulation tests and evaluations on real-world data, our proposed Bootstrapping method exhibited competitive performance compared to common DEA tools (edgeR, DESeq2, Limma-Voom). A key advantage of the Bootstrapping method is its independence from any assumptions about the sample distribution. This independence avoids the bias raised from inaccurate assumptions, offering the potential for broad application across various areas of genomic research.
Metatranscriptomic analysis of pediatric acute sinusitis: pathogen detection and host response profiling
(University of Waterloo, 2024-05-08) Abu Mazen, Nooran
Acute sinusitis (AS) is the fifth leading cause of antibiotic prescriptions in children. Distinguishing bacterial AS from common viral upper respiratory infections in children is crucial to prevent unnecessary antibiotic use but is challenging with current diagnostic methods. Despite its speed and cost, untargeted RNA sequencing (RNA-seq) of clinical samples from children with suspected AS has the potential to overcome several limitations of other methods. However, the utility of sequencing based approaches in analysis of AS has not been fully explored. Here, we performed RNA-seq of nasopharyngeal samples from 221 children with clinically diagnosed AS to characterize their pathogen and host-response profiles. Results from RNA-seq were compared with those obtained using culture for three common bacterial pathogens and qRT-PCR for 12 respiratory viruses. Metatranscriptomic pathogen detection showed high concordance with culture or qRT-PCR, showing 87%/81% sensitivity (sens) / specificity (spec) for detecting bacteria, and 86%/92% (sens/spec) for viruses, respectively. 22 additional pathogens not tested for in the clinical panel were detected, and plausible pathogens were identified in 11/19 (58%) of cases where no organism was detected by culture or qRT-PCR. 205 viruses were assembled across the samples including novel strains of coronaviruses, respiratory syncytial virus (RSV), and enterovirus D68. By analyzing host gene expression, host-response signatures were identified that distinguished bacterial and viral infections and correlated with pathogen abundance. Ultimately, this study demonstrates the potential of untargeted metatranscriptomics for in depth analysis of the etiology of AS, comprehensive host-response profiling, and using these together to work towards optimized patient care.
Metabolic engineering strategies for biomanufacturing of chemicals using Yarrowia lipolytica and Escherichia coli
(University of Waterloo, 2024-04-24) Kefale, Teshager Bitew
This dissertation advances metabolic engineering by optimizing the genetic and metabolic capabilities of Yarrowia lipolytica and Escherichia coli to enhance their applications in biotechnology. It focuses on improving Y. lipolytica's mannitol and amino acids production by varying fermentation temperatures and employing techniques like shake flask fermentation, HPLC, and NMR. Notably, mannitol production was enhanced through targeted modifications of FBP1 gene at elevated temperatures. RNAseq analyses highlighted shifts in metabolic pathways under thermal stress, markedly in lipid, sugar and amino acids metabolism. Additionally, a dual-gRNA CRISPR-Cas9 system was integrated within the pCRISPRYL2 plasmid, noticeably improving genetic editing precision by overcoming the constraints of the non-homologous end joining (NHEJ) pathway. Furthermore, the study pioneered a Cell-Free Metabolic Engineering (CFME) strategy to synthesize 5-Aminolevulinic Acid (5-ALA) utilizing optimized enzymatic reactions and operational conditions, presenting a scalable and eco-friendly alternative to conventional whole-cell systems. In parallel, engineered E. coli demonstrated robust heme production capabilities in both whole-cell and cell-free systems. Heme derivatives, including valuable pigments like biliverdin, Phycocyanobilin (PCB) and Phycoerythrobilin (PEB) were also produced at a 1L bioreactor scale utilizing E. coli engineered with unexplored enzymes. Overall, this work not only expands the scope of metabolic engineering but also sets a foundational work for future innovations in biomanufacturing.
Structural and Biochemical Insights into Previously Uncharacterized Activity of Nucleotide-Dependent Phosphoenolpyruvate Carboxykinases
(University of Waterloo, 2024-04-08) Barwell, Sarah
Phosphoenolpyruvate carboxykinase (PEPCK) enzymes are central to glucose metabolism, with their main role in catalyzing one of the first steps of gluconeogenesis. In addition to its primary role, PEPCK is also involved, whether directly or indirectly, in glyceroneogenesis, amino acid metabolism, and lipogenesis. The PEPCK enzyme is responsible for the conversion of oxaloacetic acid to phosphoenolpyruvate using a triphosphate nucleotide as a phosphoryl donor. This dissertation focuses on human cytosolic PEPCK and Escherichia coli PEPCK as representative enzymes of the PEPCK family. The hcPEPCK enzyme is well characterized and extensively studied. Recent research suggests a new role for this enzyme, and potentially other GTP-dependent PEPCKs, as a protein kinase. hcPEPCK is thought to phosphorylate the ER protein INSIG, resulting in a cascade effect leading to increased lipogenesis. Expanding upon the information in the literature, phosphomimetic mutants were used to study the effects of phosphorylation on hcPEPCK, and its subsequent interactions with the INSIG protein. The ATP-dependent PEPCKs are less studied than their GTP counterparts, and some of the research in the literature is not up to date. Historically, the bulk of the research has involved GTP-dependent PEPCK. Using E. coli PEPCK as a proxy for the ATP-PEPCK class, a full kinetic and structural characterization was performed to further our understanding on these enzymes and compare differences and similarities between the classes. In addition, a new 31P-NMR assay was designed to address the hypothesis that ATP-dependent PEPCK can catalyze two additional reactions: the conversion of phosphoenolpyruvate to pyruvate (pyruvate kinase-like activity), and the nucleotide-dependent catalysis of phosphoglycolate.
Environmental DNA barcoding as a method of amphibian species detection compared to conventional monitoring techniques in southern Ontario vernal pools
(University of Waterloo, 2024-03-06) Zamora, Cailyn
Ongoing monitoring is vital for the conservation of amphibian species and is conducted through conventional auditory and visual surveys. A molecular method, termed environmental DNA (eDNA) barcoding, may offer a more sensitive method of species detection that negates the need for direct species observation. The research aims of this thesis were to conduct a comparative analysis of eDNA barcoding versus conventional (audio/visual) species detection methods for six amphibian species in southern Ontario. I hypothesized that eDNA barcoding would offer equal or greater species presence detections compared to the conventional methods. Conventional surveys and eDNA collections were conducted in three vernal pools from April-July 2019 in collaboration with rare Charitable Research Reserve (Cambridge, ON). Conventional methods included collection of daily audio files from acoustic song meters and weekly/biweekly visual encounter surveys. Audio data was analyzed using Kaleidoscope Pro. Alongside conventional surveys, duplicate water samples containing eDNA were collected at multiple sampling locations around three vernal pools. After water collection, eDNA was concentrated by filtration, extracted, and quality controlled. eDNA samples were processed using optimized eDNA barcoding assays using quantitative PCR. Comparative analysis between conventional methods and eDNA barcoding contradicts a one-size-fits-all model of amphibian monitoring. eDNA barcoding offered a reliable and effective method of species detection for five of the target amphibians especially for obligate vernal pool breeding species, however this method failed to accurately detect the spring peeper despite detections by passive acoustic surveys. I propose using eDNA barcoding alongside a conventional method of species detection to optimize detections across a spatiotemporal scale, however, this should be catered to the target species of interest. Future studies could implement a multi-year study as well as a comparison of eDNA barcoding to metabarcoding for Ontario amphibian species. eDNA barcoding offers a new method of species detection that could aid in ongoing amphibian monitoring and therefore conservation efforts of the declining taxa.
Seasonal and environmental effects on physiological tolerances and gill adaptations of imperilled Eastern Sand Darter (Ammocrypta pellucida)
(University of Waterloo, 2024-02-15) Firth, Britney
In the absence of accurate biological information, conservation and recovery strategies carry the risk of failing to meet their objectives. Understanding species-specific physiological tolerances is important for several aspects of conservation, such as better understanding habitat requirements, determining the significance of future environmental change, and evaluating the suitability of recovery activities such as reintroduction. Currently, there is relatively little scientific information on the physiological tolerances of Eastern Sand Darter (Ammocrypta pellucida), a small benthic fish listed as threatened under the Species at Risk Act in Canada. The goal of this thesis was to define temperature and oxygen tolerances of Eastern Sand Darter and determine the role of temperature and turbidity, two common environmental stressors, on these tolerances. I also aimed to determine the response of three co-occurring darter species to the same environmental stressors. All objectives were conducted to evaluate physiological thresholds, thereby improving our understanding of species habitat, which allows us to identify restoration priorities and evaluate the suitability of candidate sites for reintroduction. To achieve this goal, field trials were conducted between June and November 2019 in the Grand River, Ontario, to encompass a range of ambient water temperatures (7-25℃) and August 2020 in the comparatively more turbid Thames River, Ontario. Critical thermal maximum (CTmax), agitation temperature (Tag), hypoxia tolerance (loss of equilibrium, LOE; and critical oxygen tension), and metabolic rate were measured to test the effect of seasonal temperature change and turbidity. Gills were collected from incidental mortalities of Eastern Sand Darter, and from three co-occurring darter species (Blackside Darter, Percina maculata; Greenside Darter, Etheostoma blennioides; and Johnny Darter, Etheostoma nigrum) to assess gill morphometrics. Chapter 2 demonstrated that Tag and CTmax significantly increased linearly with water temperature but there was no significant influence of turbidity. However, there was a significant interaction between turbidity, mass, and CTmax, indicating that turbidity has a hypothesized, indirect effect on CTmax by reducing growth. Overall, study results better define the sensitivity of Eastern Sand Darter to temperature and turbidity fluctuations and provide a better understanding of suitable thermal habitat. In Chapter 3, I demonstrated that temperature alone had no impact on metabolism but significantly and positively impacted hypoxia tolerance metrics (Loss of equilibrium and critical oxygen tensions). However, temperature explained very little of the variability seen in the response of metabolism and both hypoxia tolerance metrics across seasons, while environmental and fish-specific factors (reproduction and condition) explained more of the variation. Overall, temperature may be a weak predictor for Eastern Sand Darter metabolism and hypoxia tolerance, but the identification of a relatively high hypoxia tolerance helps to explain why the species persists in sand substrates that may have low availability of oxygen. In Chapter 4, I determined that turbidity significantly increased gill interlamellar space, filament width, and loss of equilibrium (i.e. decreased hypoxia tolerance) but had no significant influence on any other gill morphometric measurements, or on species metabolism. The findings suggest that turbidity has limited impact on Eastern Sand Darter gill physiology and that current turbidity levels do not surpass most physiological thresholds of Eastern Sand Darter. Chapter 5 demonstrated that temperature and turbidity have limited impact on the gill morphology of three common darter species, only impacting ionocyte number, lamellae width, and hematocrit. Findings suggest that the three darter species have limited plasticity in gill morphology under the range of temperature and turbidity tested. Having data on other darter species lets one put Eastern Sand Darter in context of other members of the ecosystem that are phylogenetically related. Overall, my thesis provides information on the physiological tolerances of Eastern Sand Darter that can be used to inform species recovery under Canada’s Species at Risk Act, such as refining the description of critical habitat, identifying habitat restoration objectives, and evaluating the suitability of candidate sites for reintroduction. Additionally, the discovery that current turbidity levels have limited impact on Eastern Sand Darter physiology allows for a refined understanding of how siltation impacts the species survival.
Transcriptional regulation of North American wood frog (Rana sylvatica) dorsal skin during development and in response to chronic exposure to a microplastics mixture
(University of Waterloo, 2024-02-09) Thompson, John Drew
Amphibian populations have been experiencing declines globally for decades due to factors such as habitat loss, invasive species, climate change, overexploitation, pollution, and disease. Skin is an important immune organ in amphibians, employing physical, chemical, microbiological, and immunological defences to prevent infection. Amphibian skin also undergoes dramatic morphological changes during metamorphosis. Therefore, it is important to gain a deeper understanding of the biological changes that amphibian skin undergoes during metamorphosis and whether these tightly regulated processes can be disrupted by environmental contaminants. Microplastics are a persistent, ubiquitous environmental contaminant of increasing concern with a wide range of effects on exposed organisms. Previous studies have reported mortality, reduced growth, behavioural changes, and hepatotoxicity in amphibians exposed to microplastics. However, few studies have evaluated sub-organismal effects of microplastic exposure on amphibians, particularly in relation to the immune system. Furthermore, few studies have chronically exposed amphibians to an environmentally realistic microplastics mixture. To address these gaps, an outdoor mesocosm experiment was performed where developing North American wood frogs (Rana sylvatica) were exposed from embryos throughout metamorphosis to a microplastics mixture consisting of an equal number of particles by count of polypropylene, polystyrene, and polyethylene terephthalate microplastics. Developing wood frogs were exposed to one of three concentrations: a 0× negative control of 0 g/L, a 1× treatment of 0.069 g/L, or a 10× treatment of 0.691 g/L of the microplastics mixture. This thesis reports on the transcriptomic analysis of dorsal skin of wood frogs from three different developmental timepoints: (1) after 50 days of exposure, average Gosner stage (GS) 34; (2) after 74 days of exposure, average GS 42; and (3) GS 45, 74 – 82 days of exposure. The first analysis performed compared the dorsal skin transcriptome in the 0× unexposed wood frogs at different timepoints to evaluate changes in dorsal skin gene expression during development. Trends corresponding to known developmental phenomena such as keratinization of the skin and active ion transport nearing the end of metamorphosis were observed in the data. Downregulation of immune genes was also seen in the skin after the first timepoint, and is in line with the systemic immunosuppression metamorphosing amphibians undergo leading up to and during metamorphic climax. This result demonstrates that the systemic immunosuppression is paired with suppression of immune function in skin tissues. The second analysis evaluated the effects of microplastic exposure on the dorsal skin transcriptome at each of the three selected developmental stages. Concentration- and timepoint-dependent effects were observed, with the greatest number of differentially expressed genes found in the 1× treatment at the first timepoint (GS ~34). Clusters of differentially expressed genes involved in mitochondrial function, protein synthesis, transmembrane transport, and immunity were found in at least one timepoint/treatment group. Developmental plasticity, morphological and behavioural changes during development, and a potential nonmonotonic dose response to microplastics in the wood frog are ideas proposed to account for the differences in response seen to different microplastic concentrations and at different developmental timepoints. Downregulation of immune genes was seen at the first developmental timepoint in response to the 1× microplastic treatment, suggesting that microplastics could cause developmental stage-specific immunosuppression in developing wood frogs. Because amphibian populations are already experiencing declines due to infectious diseases, it is important for future studies to continue to evaluate the potential for microplastics to act as a stressor and predispose amphibians to infection.
High-throughput OpenArray™: novel, swift TaqMan® Real-Time PCR chip for diagnostic biomarkers in Oncorhynchus tshawytscha
(University of Waterloo, 2024-01-26) Dang, Xiaoqing
In 2014, aquaculture replaced capture fisheries as the primary source of fish supply for human consumption for the first time in history, but to date, fish domestication has not developed enough to cope with the disease-related challenges. Members of the Salmonidae family are among the most popular commercialized fish species and salmon aquaculture has been the fastest growing food production system worldwide. An OpenArray™ device for Chinook Salmon (Oncorhynchus tshawytscha) health surveillance was validated in this thesis. On each microscope slide–sized OpenArray™ plate, 28 gene transcripts from 48 samples can be quantified using TaqMan® RT-qPCR. To evaluate the function of the chip, fish culture water, mucus, head kidney, spleen, and gill tissues were collected from Chinook salmon that were intraperitoneally injected with live Vibrio anguillarum within the course of 96 h. Based on the on-chip primer efficiency results, primers for 27 of the genes were considered acceptable for future validation. Based on the transcript expression patterns revealed by the OpenArray™ TaqMan® RT-qPCR chip, 6 immune genes (calm, mhc-i, mhc- ii, il-1β, il-8, tapbp) were selected and validated by SYBR RT-qPCR. Pearson’s correlation analysis revealed that a moderate to strong correlation existed between the results generated by both RT-qPCR methods for the 6 genes analysed. Both OpenArray™ TaqMan® and SYBR RT-qPCR detected significantly increased il- 1β and il-8 expression levels in spleen tissue at 72 h and 96 h, although the peaks of the expression were not recorded within the time point analysed. In head kidney tissue, il-1β expression level was also increased at 72 h, but there was no difference observed for il-8 between the control and infected groups within any time points. A longer response initiation time was observed in the Chinook salmon model in this trial. Comparing to previous studies in related salmon species, the pro-inflammatory cytokine il-1β was observed to increase as early as 6 h post infection in vivo. Nevertheless, mucus samples showed great potential as a minimally invasive sampling method if the transcripts were quantified by SYBR RT-qPCR. By comparison, in mucus the OpenArray™ TaqMan® RT-qPCR chip did not report as many quantification data as in spleen and head kidney tissue. In general, both RT-qPCR methods revealed an early systematic immune response induced by Vibrio anguillarum in spleen and head kidney, but the biological significance of the delayed immune response initiation will require further analysis to understand. The OpenArray™ TaqMan® RT-qPCR chip validated in this thesis was designed to be specific to salmon species under the order Salmoniformes. By validating this health monitoring tool in other salmon species, it is possible that the interspecies physiological differences can be horizontally compared. This will enable the development of species- specific, more effective prophylactic and therapeutic approaches against the salmon diseases.
Microbial Community Compositional Stability in Agricultural Soils During Freeze-Thaw and Fertilizer Stress
(University of Waterloo, 2024-01-22) Jensen, Grant
Microbial activity persists in cold region agricultural soils during the fall, winter, and spring (i.e., non-growing season) and frozen condition, with peak activity during thaw events. Climate change is expected to change the frequency of freeze-thaw cycles (FTC) and extreme temperature events (i.e, altered timing, extreme heat/cold events) in temperate cold regions, which may hasten microbial consumption of fall-amended fertilizers, decreasing potency come the growing season. In this thesis, I conducted a high-resolution temporal examination of the impacts of freeze-thaw and nutrient stress on microbial communities in agricultural soils across both soil depth and time. Four soil columns were incubated under a climate model of a non-growing season including precipitation, temperature, and thermal gradient with depth over 60 days. Two columns were amended with fertilizer, and two incubated as unamended soil. The impacts of repeated FTC and nutrient stress on bacterial, archaeal, and fungal soil community members were determined, providing a deeply sampled longitudinal view of soil microbial response to non-growing season conditions. Geochemical changes from flow-through leachate and amplicon sequencing of 16S and ITS rRNA genes were used to assess community response. Despite nitrification observed in fertilized columns, there were no significant microbial diversity, core community, or nitrogen cycling population trends in response to nutrient stress. FTC impacts were observable as an increase in alpha diversity during FTC. Community compositions shifted across a longer time frame than individual FTC, with bulk changes to the community in each phase of the experiment. My results demonstrate microbial community composition remains relatively stable for archaea, bacteria, and fungi through a non-growing season, independent of nutrient availability. This observation contrasts canonical thinking that FTC have significant and prolonged effects on microbial communities. In contrast to permafrost and other soils experiencing rare FTC, in temperate agricultural soils regularly experiencing such perturbations, the response to freeze-thaw and fertilizer stress may be muted by a more resilient community or be controlled at the level of gene expression rather than population turn-over. These results clarify the impacts of winter FTC on fertilizer consumption, with implications for agricultural best practices and modeling of biogeochemical cycling in agroecosystems.
Characterization of Novel Protease Flagellinolysin in Pseudoalteromonas tunicata
(University of Waterloo, 2024-01-03) Jenkins, Benjamin
Flagellinolysins are a family of novel enzymes that act both as structural flagellins and proteases. Although previously identified and shown to be both structurally integrated into filaments and proteolytically active, flagellinolysin’s biological function has remained unknown. The size of the bacterial flagellum and its prevalence in the natural world mean an enzymatic flagellin could have a significant effect in shaping the microscopic and macroscopic environments in ways that have as of yet gone undiscovered. This thesis represents a multipronged approach to characterize flagellinolysin in the marine bacterium Pseudoalteromonas tunicata, elaborate upon its context, and attempt to uncover both its role and possible substrates. The first avenue of exploration relies upon building an understanding of flagellinolysin’s context in the natural world. This includes a phylogenetic survey of flagellinolysin’s presence across the bacterial tree of life, the identification of a likely regulatory method, and its presence within a flagellar gene cluster. Flagellinolysin is also identified as being expressed through both qrtPCR and proteomics, and shown to be integrated into mature filaments by antibody-based techniques. Flagellinolysin’s nature in P. tunicata as a metalloprotease is also affirmed here by demonstrating abolition of activity through mutation. With this information in hand, two mutant strains were generated: a catalytically inert point mutant (E238A) and a whole gene knockout mutant (ΔFlaMP). These mutants were shown to be capable of motility and to produce full-length flagella capable of generating movement, but were found to also produce defective biofilms that produce hyperaggregated architecture, indicative of difficulties in spreading across surfaces. Defective biofilms led to an investigation to try and identify a potential substrate and determine a mechanism for the biofilm defect phenotype. Multiple proteomics-based methods were deployed, eventually pointing to a putative surface adhesin (VCBS) as a promising candidate. This has culminated in the development of a possible model of flagellinolysin function similar to the LapG/A system and opened the possibility of an entirely new chapter in flagellar adhesion, flagellar sheaths, and biofilm regulation.
Assessing the utility of passive sampling for building-scale SARS-CoV-2 wastewater-based surveillance to inform public health action
(University of Waterloo, 2023-12-21) Haskell, Blake
Wastewater-based surveillance (WBS) is an effective public health tool that has been used to detect human viruses for decades. Most recently it has been applied to monitor SARS-CoV-2 RNA in municipal wastewater to track the prevalence and spread of COVID-19 in communities during the pandemic. Much of this work has been performed at the wastewater treatment plant (WWTP) prior to treatment, where it has been shown that WBS is closely related to clinical infections and hospitalizations. The application of WBS has gradually expanded to include upstream sites within the sewershed where neighbourhood and building-scale surveillance can be performed. However, sampling in these upstream environments introduces additional challenges for sampling and interpretation. The intermittent flow and composition of wastewater in the sewers close to the source greatly influences the variability and ability to accurately detect and quantitate the target viral fragments. One approach to circumvent some of these challenges is to employ a passive sampling approach where a chosen material is immersed in the sampled medium and left to passively accumulate a target analyte of interest over time. The sampling material is consistently exposed to the sampling environment which may reduce the likelihood of false negative detections. In this thesis, a two-tiered, trigger-based wastewater surveillance program was developed on the University of Waterloo (Waterloo, Ontario) campus residences during an active public health emergency (COVID-19 pandemic). The objective was to determine if WBS using passive sampling can be used to inform institution-level public health action. Preliminary pilot studies validated a passive sampling method capable of detecting SARS-CoV-2 RNA in a municipal sewage system. Three candidate materials held in plastic frames (e.g., torpedo shaped perforated tubes) were tested for their efficacy in this application, including electronegative membrane filters and standard tampons. Cotton gauze was selected as the sampling medium for routine surveillance as it was able to detect the virus the most consistently and retained more suspended solids than the other materials tested. Twenty-four-hour passive samples were then collected three days per week over an eight-month surveillance period at selected utility holes associated with residences on the University of Waterloo campus. Two nucleocapsid gene targets (N1 and N2) of SARS-CoV-2 as well as the endogenous fecal indicator pepper mild mottle virus (PMMoV) were washed from the samplers, concentrated, extracted, and then quantified using real-time quantitative polymerase chain reaction (RT-qPCR). PMMoV is an endogenous indicator associated with human feces which has been used to normalize SARS-CoV-2 concentrations and account for dilution effects. The SARS-CoV-2 results were reported to the University health team in near real time (<12 h from sample collection). The developed workflow prioritized surveillance population coverage and minimal resource usage to address a variety of complex stakeholder needs and best support public health decision-making. The period of the study included two contrasting exposure scenarios prior to and after the rapid emergence of the SARS-CoV-2 Omicron (B.1.1.529) variant. In the fall of 2021, community viral burden was low and a tiered sampling network was able to isolate individual clinical cases at the building-scale. In the winter of 2022 wastewater signals were quickly elevated by the emergence of the highly transmissible Omicron variant. The high prevalence of SARS-CoV-2 shifted surveillance objectives from isolating cases to monitoring trends. Throughout the surveillance period, comparisons between detection results and reported clinical cases revealed that passive samplers positively identified all but one of 203 infected individuals over eight months. In one instance surveillance led to the pre-symptomatic detection of a single individual at a site monitoring over 1300 students. WBS detected the infected individual two days in advance of clinical testing confirmation, demonstrating the efficacy of the tiered passive sampling approach in supporting public health action. Remarkably, SARS-CoV-2 concentrations on passive samplers were significantly correlated with confirmed clinical cases within the upstream sewershed. The strongest correlations were observed when clinical cases were assumed to have been shedding for 10 days from reporting illness. Additionally, comparisons between SARS-CoV-2 concentrations detected in campus sewers and in municipal wastewater influent suggest that the spread of COVID-19 on the campus was similar to that of the broader community. Periods of increasing and decreasing viral burden were captured at both sampling scales and closely mirrored each other in winter 2022. These results add to the mounting evidence that passive samplers are capable of producing semi-quantitative data that reflects the prevalence of disease within the sewer catchment. Alongside routine surveillance, methodological refinement occurred in parallel with routine surveillance efforts with the goal of maximizing data insight and actionability. This included routinely evaluating samples for evidence of RT-qPCR inhibition which saw a marked increase when students returned to campus and wastewater production increased. Modifications to the workflow were made to reduce inhibition and increase confidence in surveillance results. PMMoV concentrations on passive samplers were not reflective of upstream population differences and normalization of SARS-CoV-2 with PMMoV did not improve correlations with a clinical dataset. The results suggest that saturation of the material occurred during the exposure period thus limiting the utility of PMMoV as a normalizer to account for dilution effects. This investigation demonstrates that passive sampling can be used as an effective tool to guide highly localized public health action. Spatially refined wastewater surveillance can support pandemic management decision making by acting as an early warning system, providing a basis for targeted health intervention and possible clinical testing, and is able to track spatiotemporal variations in viral RNA concentrations. The utility of a tiered surveillance approach described in this thesis demonstrates the importance of developing versatile methodologies that can be applied at varying spatial scales to address emerging public health challenges.
Analysis of Cyanobacteria and its Abundance in the Turkey Lakes Watershed using Molecular Techniques
(University of Waterloo, 2023-12-21) Krishna, Anjali
Toxic cyanobacterial blooms continue to pose a threat to the quality and safety of drinking water globally by producing toxins and forming dense surface blooms. Forested watersheds naturally provide high quality drinking water to various communities but are threatened by bloom events that are increasing due to warming climates and anthropogenic land use. Monitoring programs utilized in drinking water sources are required to adapt to the changing intensity and frequency of these blooms where observation of cyanobacterial composition and abundance may vary based on sampling efforts. However, due to the variation and adaptability of these organisms spatially and temporally, cyanobacteria are often overlooked if surface blooms are not visualized, where these organisms may be present and abundant throughout the water column at different depths and vary throughout the day. The undetected organisms may release potent toxins that are threats to drinking water security if left untreated. The harmful toxic blooms comprise of cyclic hepatotoxins, involved in causing severe liver damage and affecting human and aquatic health. The aim of this study was to identify and quantify the cyanobacterial community composition and abundance of potential toxin producing genes in an oligotrophic northern forested watershed (Turkey Lakes Watershed, Ontario, Canada). To evaluate the composition and abundance, water samples were collected from Little Turkey Lake in May, June, July, and August 2022 at integrated and varying depths to determine variability over a summer season and at different timepoints in a single day. Microbial DNA was extracted from the water samples for 16S rRNA gene sequencing where data was obtained for bioinformatic and phylogenetic analyses. Extracted samples underwent quantitative PCR analysis for identification of gene copy numbers of cyanobacteria and potential microcystin producing organisms. With the extension of the ice-free season through warmer temperatures, and changes in environmental parameters, cyanobacteria and potential cyanotoxin producers appeared as early as May in this oligotrophic lake system. Peak abundances of cyanobacterial and potential cyanotoxin producing gene copy numbers were observed in the months of July and August, without visible blooms during sampling. Cyanobacterial composition had variability between the months, days, and timepoints when sampling, demonstrating the importance of consisting monitoring and sampling efforts due to the changing composition and abundance. Variation was observed among the depths within the water column, where integrated sampling provided a snapshot of the water system and can be useful for efficient analysis of the system, but multiple depth sampling is more representative of the community composition and abundance of cyanobacteria. This illustrates that monitoring protocols for drinking water sources require evaluation for the appropriate sampling protocol, timepoints, and location of the water column as each water system is unique. This research provides insight into cyanobacterial emergence in earlier summer months in an oligotrophic water system. It is applicable for the development of monitoring and drinking water treatment protocols for toxin-producing cyanobacteria, where analysis of the full water column is required with consistent sampling and integrated sampling is efficient, especially when there is an absence of a visible surface bloom. The inclusion of molecular characterization (amplicon sequencing and qPCR) is a valuable tool that can be cost efficient and effective ways to analyze samples. This research can then be expanded to other toxins and secondary metabolites produced by cyanobacteria.
Comparative analysis and visualization of microbial gene neighborhoods: applications to pathogen genomics
(University of Waterloo, 2023-12-20) Wei, Xin
Gene neighborhoods are clusters of genes that are encoded together within the same genomic region, and may share similarity in expression and/or function. Bioinformatic analysis of gene neighborhoods is a powerful approach that provides insights into genome structure, function, and evolution. Despite its importance, existing bioinformatics tools are limited in terms of their ability to analyze and compare gene neighborhoods, especially on a large-scale. In this thesis, I present a novel software tool called AnnoView for large-scale exploration and analysis of microbial gene neighborhoods. I then demonstrate its use by exploring several different bacterial pathogens and their virulence factors, and using genomic context analysis to gain novel insights into pathogen evolution and genome function. AnnoView is a new gene neighborhood analysis tool that facilitates interactive explo- ration of microbial gene neighborhoods from 30,238 bacterial genomes and 1,672 archaeal genomes with pre-computed functional annotations derived from KEGG, Pfam, and TIGR- FAM. Users also have the flexibility to upload custom datasets in various formats for gene neighborhood visualization. As a first application of AnnoView, I analyzed the adenylate isopentenyl transferase (IPT) gene in bacteria, which encodes a cytokinin-producing enzyme found in plant pathogenic as well as plant growth-promoting bacteria (PGPB). To understand how this gene may function differently between pathogens and PGPB, AnnoView was used to explore and compare its genomic contexts across bacteria. Analysis revealed numerous distinguishing features, including the tendency for pathogen-associated adenylate IPT genes tend to occur in predicted virulence loci. As a second case study utilizing AnnoView, two novel gene clusters were discovered con- taining putative clostridial neurotoxin (CNT) genes within the genomes of Paraclostridium ghonii and Bacillus toyonensis. Both gene clusters were analyzed using AnnoView and found to contain unique features not present in other CNT-containing gene clusters. In particular, analysis of the P. ghonii toxin gene neighborhood revealed the identification of nearby genes indicating that P. ghonii toxin may have a specificity toward insect hosts. This prediction was experimentally validated in collaborative work, revealing P. ghonii toxin as a novel insecticidal neurotoxin. Lastly, as a third case study, I used AnnoView to perform a comparative genomic analysis of a putatively novel species of Clostridium known as ”clade X” identified from ancient DNA. The identification of conserved, unique gene neighborhood patterns within clade X but absent from closely related Clostridium genomes reinforces previous claims that clade X represents a distinct species. Ultimately, comparative analysis of gene neighborhoods using AnnoView and large-scale genomic information from existing databases (GTDB, AnnoTree, NCBI) is a powerful approach for for microbial genomics studies. Comparative analysis of gene neighborhoods in pathogenic genomes helps to expand our knowledge of virulence factors and pathogen- associated genomic traits, contributing to the characteristic of pathogen biology including even the identification of host species.
Application of Environmental Metabolomics for Assessment of Aquatic Invertebrate Sensitivity to Naphthenic Acids
(University of Waterloo, 2023-12-14) Johnsen, Philip
Naphthenic acids (NAs) are a class of chemicals found in oil sands process waters (OSPW) from the extraction of bitumen from surface mined oil sand. In Alberta, Canada, OSPW is currently stored indefinitely in tailing ponds and there are environmental concerns about seepage or spillage. NAs are known to be toxic to aquatic organisms. However, NA toxicity data are limited to primarily acute effects in a few species. Due to difficulties in comparing NA toxicity results, an extracted solution referred to as naphthenic acid fraction components (NAFCs) can be used as it is representative of OSPW toxicity and is well characterized. The goal of this research is to inform NA standards by performing chronic toxicity tests to derive metabolomic responses and estimates of survival. I exposed the ramshorn snail (Planorbarius corneus), and nymphs of two dragonflies, the common whitetail (Plathemis lydia) and the eastern pondhawk (Erythemis spp.), to 0, 6, 12, and 25 mg/L NAFCs for 21-days (snails) or 14-days (dragonflies) in laboratory microcosm experiments. Survival was altered only in the common whitetail with NAFC exposure. Snails laid approximately double the number of egg masses at higher concentrations of NAFCs. The metabolome responded differently among taxa. NAFC exposure altered the metabolome of only the common whitetail. Metabolite analysis for the common whitetail found that most altered metabolites were amino acids. The identified metabolites were involved primarily in pathways related to energy metabolism and protection from oxidative stress. My findings indicate that sensitivity to NAFCs is taxon-specific and demonstrate that an organism’s metabolome can provide insight into toxic effects of NAFCs.
Algal priming mediates the effects of light and nutrients on organic matter processing: insights from artificial and natural streams
(University of Waterloo, 2023-10-31) Pinks, Lauryn
Environmental factors such as light and nutrients may play important roles in determining detrital decomposition through their effects on primary production. Thus, human activities that alter the availabilities of light and nutrients may have direct consequences on organic matter (OM) processing and nutrient cycling in freshwater ecosystems. I compared heterotrophic function (OM processing and respiration) between two P levels (5 µg P L-1 and 51 µg P L-1) and two shade levels (0% and 80% shade) in natural streams, as well as 3 P levels (10 µg P L-1, 50 µg P L-1 and 100 µg P L-1) and 4 shade levels (0%, 50%, 80% and 100%) in artificial streams using the cotton-strip assay (CSA). Data from these experiments show a negative association between algal abundance (GPP and chlorophyll a) and recalcitrant OM (ROM) processing, implying a negative priming effect. Light was an important driver of negative priming which disproportionately affected ROM decomposition at lower P treatments over time. Overall, there were limited interactive effects between light and nutrient availability; rather, ROM processing was positively associated with P availability at all light levels while it was negatively associated with light availability at all P levels. There was no evidence to support positive priming (i.e., algal stimulation of ROM processing) in either experiment. Overall, results from this study illustrate the importance of considering light levels and nutrient availability when considering long term C and nutrient budgets in freshwater ecosystems.

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